Cell adhesion generally involves recognition of specific adhesive proteins by cell surface receptors. A family of cell surface receptors of particular interest to the present invention are the Integrins.
According to Hynes, Cell, 48:549-554 (1987), Integrins are a functionally and structurally related group of receptors that interact with a wide variety of ligands including extracellular matrix glycoproteins, complement and other cells. Integrins participate in cell-matrix and cell-cell adhesion in many physiologically important processes including embryological development, hemostasis, thrombosis, wound healing, immune and nonimmune defense mechanisms and oncogenic transformation. At least two human genetic diseases, Glanzmann's thrombasthenia and leukocyte adhesion deficiency, involve members of the Integrin family.
Structurally, Integrins are heterodimeric complexes comprised of noncovalently associated alpha and beta subunits. Within the Integrin family there are recognized subfamilies related by the presence of a similar beta subunit and members within each group are distinguished by distinct alpha subunits.
For instance, recent evidence indicates that an Integrin found on the surface of platelets and known as GPIIb-IIIa is one of several adhesion receptors that have distinct alpha subunits but share a similar beta subunit and the functional property of recognizing the tripeptide amino acid residue sequence Arg-Gly-Asp (using single letter symbols, RGD). Pytela et al., Science, 231:1559-1562 (1986) and Ruoslahti et al., Cell, 44:517-518 (1986). In addition to GPIIb-IIIa, this group of related receptors includes the vitronectin receptor (VnR) and fibronectin receptor (FnR) isolated from osteosarcoma cells. Pytela et al., Cell, 40:191-198 (1985), and Pytela et al., Proc. Natl. Acad. Sci. USA, 82:5766-5770 (1985).
The similar functional, structural, and antigenic properties of these proteins suggests GPIIb-IIIa and VnR are members of an Integrin subfamily for which the designation "cytoadhesin" has been proposed. Plow et al., Proc. Natl. Acad. Sci. USA. 83:6002-6006 (1986). Within the cytoadhesin group, distinct alpha subunits combine with a common or very similar beta subunit, resulting in functionally distinguishable receptors. Ginsberg et
al., J. Biol. Chem., 262:5437-5440 (1987).
For example, GPIIb-IIIa is a heterodimer complex comprised of alpha and beta subunits. Jennings et al., J. Biol. Chem., 257:10458-10466 (1982). The alpha subunit, GPIIb, consists of a heavy chain and a light chain that are linked together by disulfide bonds. The beta subunit, GPIIIa is a single chain polypeptide of about 100 kDa. Phillips et al., J. Biol. Chem., 252:2121-2126 (1977). Cell surface molecules immunologically related to GPIIb-IIIa have been identified on a variety of cell types. See Thiagarajan et al., J. Clin. Invest., 75:896-901 (1985); Plow et al., Proc. Natl. Acad. Sci. USA. 83:6002-6006 (1986); and Fitzgerald et al., J. Biol. Chem., 260:10893-10896 (1985).
GPIIb-IIIa contributes to platelet function through interactions with RGD-containing proteins, i.e., proteins containing an Arg-Gly-Asp amino acid residue sequence, such as fibrinogen [Bennett et al., Proc. Natl. Acad. Sci. USA, 80:2417-2421 (1983)], fibronectin [Ginsberg et al., J. Clin. Invest., 71:619-624 (1983)], and von Willebrand factor [Ruggeri et al., Proc. Natl. Acad. Sci. USA, 79:6038-6041 (1982)], and therefore is a component of the common platelet adhesive protein receptor [Pytela et al., Science, 231:1559-1562 (1986) and Plow et al., J. Biol. Chem., 259:5388-5391 (1984)].
At least 2 other groups of heterodimeric adhesion receptors have been identified in which a common beta subunit combines with a number of distinct alpha subunits. One group is found on leukocytes and has been referred to as the leukocyte adhesion (LeuCam) family and includes LFA-1, Mac-1, and P150,95. Sanchez-Madrid et al., J. Exp. Med., 158:1785-1803 (1983) and Springer et al., Ciba. Found. Symp., 118:102-126 (1986). The other group is more widely distributed and has been referred to as the VLA family. Hemler et al., J. Biol. Chem., 262:3300-3309 (1987). The beta subunit of the VLA family [Hemler et al., J. Biol. Chem., 262:3300-3309 (1987)] in the chicken has been cloned, sequenced and designated "Integrin" [Tamkun et al., Cell, 46:271-282 (1986)]. The sequence of chicken Integrin is similar to that of GPIIIa [Fitzgerald et al., J. Biol. Chem., 262:3936-3939 (1987)] and to the beta subunit of the leukocyte adhesion family [Kishimoto et al., Cell, 48:681-690 (1987)]. Moreover, partial sequences of several alpha subunits also indicate similarities. Ginsberg, et al., J. Biol. Chem., 262:5437-5440 (1987); Suzuki, et al., Proc. Natl. Acad. Sci. USA, 83:8614-8618 (1986); and Charo, et al., Proc. Natl. Acad. Sci. USA, 83:8351-8356 (1986).
The sites on GPIIb-IIIa, or the other cytoadhesins, that are crucial for their functions as adhesion receptors are not presently well characterized. Several observations suggest that a functionally significant site on GPIIb-IIIa is near the epitope defined by the monoclonal antibody PMI-1. This antibody binds to the heavy chain of GPIIb [Shadle et al., J. Cell. Biol., 99:2056-2060 (1984)] and defines a region of GPIIb that is associated with several distinct functional activities. For instance, PMI-1 inhibits adhesion of washed platelets to collagen. Shadle et al., J. Cell. Biol., 99:2056-2060 (1984).
Recently, the site on the GPIIIa subunit for interaction with the RGD-containing region of fibrinogen was localized to residues 109-171 of GPIIIa by chemical crosslinking between GPIIIa and the synthetic polypeptide KYGRGDS. D'Souza et al., Science, 242:91-93 (1988). Studies to identify the site of interaction between fibrinogen gamma chain polypeptides and GPIIb-IIIa have shown an interaction with the GPIIb subunit. Kloczewick, M., et al., J. Biochem., 23:1767-74 (1984). Only recently have peptides containing the gamma chain of fibrinogen been selectively crosslinked to an identifiable region, residues 294-314, of GPIIb. D'Souza, S. E., et al., J. Biol. Chem., 265:3440-46 (1990). These results suggest a proxinal relationship between the 294-314 residue region and the ligand binding site of GPIIb-IIIa.